Endoplasmic reticulum (ER) stress elicits the unfolded protein response (UPR), initially aimed at coping with the stress, but triggering cell death upon further stress. ER stress induces the C/EBP-® variant Liver-enriched Activating Protein (LAP), followed by the dominant-negative variant, Liver Inhibitory Protein (LIP). However, the distinct role of LAP and LIP in ER stress is unknown. We found that the kinetics of the ER stress-induced expression of LIP overlapped with that of the cell death in mouse B16 melanoma cells. Furthermore, inducible over-expression of LIP augmented ER stress-triggered cell death whereas over-expression of LAP attenuated cell death. Similar results were obtained in human 293T cells. Limited vasculature in tumors triggers hypoxia, nutrient shortage and accumulation of toxic metabolites, all of which eliciting continuous ER stress. We found that LAP promoted and LIP inhibited B16 melanoma tumor progression without affecting angiogenesis or accelerating the cell cycle. Rather, LAP attenuated, whereas LIP augmented tumor ER stress. We therefore suggest that C/EBP-® regulates the transition from the protective to the death–promoting phase of the UPR. We further suggest that the over-expression of LAP observed in many solid tumors promotes tumor progression by attenuating ER stress–triggered tumor cell death.
The aqueous extract of Anemarrhena asphodeloides (BN108) induces apoptosis in various cancer cell lines but is significantly less cytotoxic in non-transformed cells. Chemical fractionation of BN108 showed that its cytotoxicity is associated with timosaponins, steroidal saponins of coprostane type. Timosaponin BII (TBII) is a major saponin in BN108, but it shows little cytotoxicity. A much less abundant TAIII induces cell death in tumor cells but not in normal cells, reproducing the selectivity of the total extract BN108. Glycosidase treatment, by removing the extra sugar moiety in TBII, converts it to TAIII and confers cytotoxic activity. Analysis of the mechanisms of death induced by TAIII revealed activation of two distinct pro-apoptotic pathways: first, inhibition of mTORC1 manifested in much reduced phosphorylation of mTORC1 targets; second, induction of endoplasmic reticulum stress culminating in phosphorylation of eIF2α and activation of caspase 4. These pro-apoptotic pathways are activated by TAIII selectively in tumor cells but not in normal cells. Both pathways play a causative role in TAIII cytotoxicity, as restoration of either mTOR activity or relief of ER stress alone offer only partial protection from TAIII. Inhibition of mTORC1 and induction of ER stress apparently contribute to the induction of the previously reported autophagic response in TAIII-treated cells. TAIII induced autophagy plays a protective role in TAIII induced death signaling, and failure to mount autophagic response is associated with heightened sensitivity to TAIII induced apoptosis. The multiple death-promoting and apparently tumor-selective responses to TAIII, its ability to inhibit mTORC1, and the possibility of further enhancing its cytotoxicity by pharmacological inhibition of autophagy, make TAIII an attractive candidate for development as a cancer therapeutic agent.
Hepatitis C virus (HCV) has evolved complex strategies to evade host immune responses and establish chronic infection. The only treatment available for HCV infections, alpha interferon (IFN-α), is effective in a limited percentage of patients. The mechanisms by which IFN-α interferes with the HCV life cycle and the reasons for limited effectiveness of IFN-α therapy have not yet been fully elucidated. Using a cell-based HCV replication system and specific kinase inhibitors, we examined the role played by various signaling pathways in the IFN-α-mediated HCV clearance. We reported that conventional protein kinase C (cPKC) activity is important for the effectiveness of IFN-α treatment. In cells treated with a cPKC-specific inhibitor, IFN-α failed to induce an efficient HCV RNA degradation. The lack of cPKC activity leads to a broad reduction of IFN-α-stimulated gene expression due to a significant impairment of STAT1 and STAT3 tyrosine phosphorylation. Thus, modulation of cPKC function by either host or viral factors could influence the positive outcome of IFN-α-mediated antiviral therapies.
Phosphorylation at a highly conserved serine residue (Ser-10) in the histone H3 tail is considered to be a crucial event for the onset of mitosis. This modification appears early in the G2 phase within pericentromeric heterochromatin and spreads in an ordered fashion coincident with mitotic chromosome condensation. Mutation of Ser-10 is essential in Tetrahymena, since it results in abnormal chromosome segregation and extensive chromosome loss during mitosis and meiosis, establishing a strong link between signaling and chromosome dynamics. Although mitotic H3 phosphorylation has been long recognized, the transduction routes and the identity of the protein kinases involved have been elusive. Here we show that the expression of Aurora-A and Aurora-B, two kinases of the Aurora/AIK family, is tightly coordinated with H3 phosphorylation during the G2/M transition. During the G2 phase, the Aurora-A kinase is coexpressed while the Aurora-B kinase colocalizes with phosphorylated histone H3. At prophase and metaphase, Aurora-A is highly localized in the centrosomic region and in the spindle poles while Aurora-B is present in the centromeric region concurrent with H3 phosphorylation, to then translocate by cytokinesis to the midbody region. Both Aurora-A and Aurora-B proteins physically interact with the H3 tail and efficiently phosphorylate Ser10 both in vitro and in vivo, even if Aurora-A appears to be a better H3 kinase than Aurora-B. Since Aurora-A and Aurora-B are known to be overexpressed in a variety of human cancers, our findings provide an attractive link between cell transformation, chromatin modifications and a specific kinase system.
Transcription factors of the CREB family control the expression of a large number of genes in response to various signaling pathways. Regulation mediated by members of the CREB family has been linked to various physiological functions. Classically, activation by CREB is known to occur upon phosphorylation at an essential regulatory site (Ser133 in CREB) and the subsequent interaction with the ubiquitous coactivator CREB-binding protein (CBP). However, the mechanism by which selectivity is achieved in the identification of target genes, as well as the routes adopted to ensure tissue-specific activation, remains unrecognized. We have recently described the first tissue-specific coactivator of CREB family transcription factors, ACT (activator of CREM in testis). ACT is a LIM-only protein which associates with CREM in male germ cells and provides an activation function which is independent of phosphorylation and CBP. Here we characterize a family of LIM-only proteins which share common structural organization with ACT. These are referred to as four-and-a-half-LIM-domain (FHL) proteins and display tissue-specific and developmentally regulated expression. FHL proteins display different degrees of intrinsic activation potential. They provide powerful activation function to both CREB and CREM when coexpressed either in yeast or in mammalian cells, specific combinations eliciting selective activation. Deletion analysis of the ACT protein shows that the activation function depends on specific arrangements of the LIM domains, which are essential for both transactivation and interaction properties. This study uncovers the existence of a family of tissue-specific coactivators that operate through novel, CBP-independent routes to elicit transcriptional activation by CREB and CREM. The future identification of additional partners of FHL proteins is likely to reveal unappreciated aspects of tissue-specific transcriptional regulation.
It is commonly accepted that pathways that regulate proliferation/differentiation processes, if altered in their normal interplay, can lead to the induction of programmed cell death. In a previous work we reported that Polyoma virus Large Tumor antigen (PyLT) interferes with in vitro terminal differentiation of skeletal myoblasts by binding and inactivating the retinoblastoma antioncogene product. This inhibition occurs after the activation of some early steps of the myogenic program. In the present work we report that myoblasts expressing wild-type PyLT, when subjected to differentiation stimuli, undergo cell death and that this cell death can be defined as apoptosis. Apoptosis in PyLT-expressing myoblasts starts after growth factors removal, is promoted by cell confluence, and is temporally correlated with the expression of early markers of myogenic differentiation. The block of the initial events of myogenesis by transforming growth factor β or basic fibroblast growth factor prevents PyLT-induced apoptosis, while the acceleration of this process by the overexpression of the muscle-regulatory factor MyoD further increases cell death in this system. MyoD can induce PyLT-expressing myoblasts to accumulate RB, p21, and muscle- specific genes but is unable to induce G00 arrest. Several markers of different phases of the cell cycle, such as cyclin A, cdk-2, and cdc-2, fail to be down-regulated, indicating the occurrence of cell cycle progression. It has been frequently suggested that apoptosis can result from an unbalanced cell cycle progression in the presence of a contrasting signal, such as growth factor deprivation. Our data involve differentiation pathways, as a further contrasting signal, in the generation of this conflict during myoblast cell apoptosis.
The phosphatidylinositol 3-kinase/AKT/mammalian target of rapamycin (PI3K/AKT/mTOR) pathway promotes melanoma tumor growth and survival while suppressing autophagy, a catabolic process through which cells collect and recycle cellular components to sustain energy homeostasis in starvation. Conversely, inhibitors of the PI3K/AKT/mTOR pathway, in particular the mTOR inhibitor temsirolimus (CCI-779), induce autophagy, which can promote tumor survival and thus, these agents potentially limit their own efficacy. We hypothesized that inhibition of autophagy in combination with mTOR inhibition would block this tumor survival mechanism and hence improve the cytotoxicity of mTOR inhibitors in melanoma. Here we found that melanoma cell lines of multiple genotypes exhibit high basal levels of autophagy. Knockdown of expression of the essential autophagy gene product ATG7 resulted in cell death, indicating that survival of melanoma cells is autophagy-dependent. We also found that the lysosomotropic agent and autophagy inhibitor hydroxychloroquine (HCQ) synergizes with CCI-779 and led to melanoma cell death via apoptosis. Combination treatment with CCI-779 and HCQ suppressed melanoma growth and induced cell death both in 3-dimensional (3D) spheroid cultures and in tumor xenografts. These data suggest that coordinate inhibition of the mTOR and autophagy pathways promotes apoptosis and could be a new therapeutic paradigm for the treatment of melanoma.
The control of cell death is a biological process essential for proper development, and for preventing devastating pathologies like cancer and neurodegeneration. On the other hand, autophagy regulation is essential for protein and organelle degradation, and its dysfunction is associated with overlapping pathologies like cancer and neurodegeneration, but also for microbial infection and aging. In the present report we show that two evolutionarily unrelated receptors—Neurokinin 1 Receptor (NK1R,) a G-protein coupled receptor, and Insulin-like Growth Factor 1 Receptor (IGF1R), a tyrosine kinase receptor—both induce non-apoptotic cell death with autophagic features and requiring the activity of the autophagic core machinery proteins PI3K-III, Beclin-1 and Atg7. Remarkably, this form of cell death occurs in apoptosis-competent cells. The signal transduction pathways engaged by these receptors both converged on the activation of the nuclear receptor NR4A1, which has previously been shown to play a critical role in some paradigms of apoptosis and in NK1R-induced cell death. The activity of NR4A1 was necessary for IGF1R-induced cell death, as well as for a canonical model of cell death by autophagy induced by the presence of a pan-caspase inhibitor, suggesting that NR4A1 is a general modulator of this kind of cell death. During cell death by autophagy, NR4A1 was transcriptionally competent, even though a fraction of it was present in the cytoplasm. Interestingly, NR4A1 interacts with the tumor suppressor p53 but not with Beclin-1 complex. Therefore the mechanism to promote cell death by autophagy might involve regulation of gene expression, as well as protein interactions. Understanding the molecular basis of autophagy and cell death mediation by NR4A1, should provide novel insights and targets for therapeutic intervention.
Topotecan produces DNA damage that induces autophagy in cancer cells. In this study, sensitising topotecan to colon cancer cells with different P53 status via modulation of autophagy was examined.
The DNA damage induced by topotecan treatment resulted in cytoprotective autophagy in colon cancer cells with wild-type p53. However, in cells with mutant p53 or p53 knockout, treatment with topotecan induced autophagy-associated cell death. In wild-type p53 colon cancer cells, topotecan treatment activated p53, upregulated the expression of sestrin 2, induced the phosphorylation of the AMPKα subunit at Thr172, and inhibited the mTORC1 pathway. Furthermore, the inhibition of autophagy enhanced the anti-tumour effect of topotecan treatment in wild-type p53 colon cancer cells but alleviated the anti-tumour effect of topotecan treatment in p53 knockout cells in vivo.
These results imply that the wild-type p53-dependent induction of cytoprotective autophagy is one of the cellular responses that determines the cellular sensitivity to the DNA-damaging drug topotecan. Therefore, our study provides a potential therapeutic strategy that utilises a combination of DNA-damaging agents and autophagy inhibitors for the treatment of colon cancer with wild-type p53.
mTOR inhibitors are used clinically to treat renal cancer but are not curative. Here we show that autophagy is a resistance mechanism of human renal cell carcinoma (RCC) cell lines to mTOR inhibitors. RCC cell lines have high basal autophagy that is required for survival to mTOR inhibition. In RCC4 cells, inhibition of mTOR with CCI-779 stimulates autophagy and eliminates RIP kinases (RIPKs) and this is blocked by autophagy inhibition, which induces RIPK- and ROS-dependent necroptosis in vitro and suppresses xenograft growth. Autophagy of mitochondria is required for cell survival since mTOR inhibition turns off Nrf2 antioxidant defense. Thus, coordinate mTOR and autophagy inhibition leads to an imbalance between ROS production and defense, causing necroptosis that may enhance cancer treatment efficacy.
Contact with HIV-1 envelope protein elicits release of ATP through pannexin-1 channels on target cells; by activating purinergic receptors and Pyk2 kinase in target cells, this extracellular ATP boosts HIV-1 infectivity.
Extracellular adenosine triphosphate (ATP) can activate purinergic receptors of the plasma membrane and modulate multiple cellular functions. We report that ATP is released from HIV-1 target cells through pannexin-1 channels upon interaction between the HIV-1 envelope protein and specific target cell receptors. Extracellular ATP then acts on purinergic receptors, including P2Y2, to activate proline-rich tyrosine kinase 2 (Pyk2) kinase and transient plasma membrane depolarization, which in turn stimulate fusion between Env-expressing membranes and membranes containing CD4 plus appropriate chemokine co-receptors. Inhibition of any of the constituents of this cascade (pannexin-1, ATP, P2Y2, and Pyk2) impairs the replication of HIV-1 mutant viruses that are resistant to conventional antiretroviral agents. Altogether, our results reveal a novel signaling pathway involved in the early steps of HIV-1 infection that may be targeted with new therapeutic approaches.
The mechanisms underlying adenovirus-mediated autophagy are currently unknown. Recently, members of the Bcl-2 protein family have been associated with autophagy. It was also reported that the Bcl-2 homology-3 (BH3) domain encompassed by both Beclin 1 and Bcl-2-like proteins is essential for their pro-autophagy or anti-autophagy functions. Here, we report for the first time that E1B19K, the adenovirus BH3 domain protein, interacts with Beclin 1 to initiate autophagy. Using immunoprecipitation assays we showed that expression of E1B19K in the host cell disrupted the physical interactions between Beclin 1 and Bcl-2 proteins. The displacement of Bcl-2 was coincident with the recruitment of PI3KC3 to the Beclin 1/E1B19K complexes. As a result of the changes in the components of the Beclin 1 interactome, there was activation of PI3KC3, as showed by the identification of PI3K-mediated lipid phosphorylation, and subsequent formation of autophagosomes. Importantly, the BH3 functional domain of E1B19K protein was required for the heterodimerization with Beclin 1. We also showed that transfer of E1B19K was sufficient to trigger autophagy in cancer cells. Consistent with these data, mutant adenoviruses encompassing a deletion of the E1B19K gene produced a marked deficiency in the capability of the virus to induce autophagy as showed by examining the lipidation and cleavage of LC3-I as well as the subcellular localization of LC3-II, the decrease in the levels of p62, and the formation of autophagosomes. Our work offers new information on the mechanisms of action of the adenoviral E1B19K protein as partner of Beclin 1 and positive regulator of autophagy.
Nonalcoholic fatty liver disease (NAFLD) is a known outcome of hepatosteatosis. Free fatty acids (FFA) induce the unfolded protein response (UPR) or endoplasmic reticulum (ER) stress that may induce apoptosis. Recent data indicate ER stress to be a major player in the progression of fatty liver to more aggressive lesions. Autophagy on the other hand has been demonstrated to be protective against ER stress- induced cell death. We hypothesized that exendin-4 (GLP-1 analog) treatment of fat loaded hepatocytes can reduce steatosis by autophagy which leads to reduced ER stress-related hepatocyte apoptosis.
Primary human hepatocytes were loaded with saturated, cis- and trans-unsaturated fatty acids (palmitic, oleic and elaidic acid respectively). Steatosis, induced with all three fatty acids, was significantly resolved after exendin-4 treatment. Exendin-4 sustained levels of GRP78 expression in fat-loaded cells when compared to untreated fat-loaded cells alone. In contrast, CHOP (C/EBP homologous protein); the penultimate protein that leads to ER stress-related cell death was significantly decreased by exendin-4 in hepatocytes loaded with fatty acids. Finally, exendin-4 in fat loaded hepatocytes clearly promoted gene products associated with macroautophagy as measured by enhanced production of both Beclin-1 and LC3B-II, markers for autophagy; and visualized by transmission electron microscopy (TEM). Similar observations were made in mouse liver lysates after mice were fed with high fat high fructose diet and treated with a long acting GLP-1 receptor agonist, liraglutide.
GLP-1 proteins appear to protect hepatocytes from fatty acid-related death by prohibition of a dysfunctional ER stress response; and reduce fatty acid accumulation, by activation of both macro-and chaperone-mediated autophagy. These findings provide a novel role for GLP-1 proteins in halting the progression of more aggressive lesions from underlying steatosis in humans afflicted with NAFLD.
Prognosis of patients with glioblastoma (GBM) remains very poor, thus making the development of new drugs urgent. Resveratrol (Rsv) is a natural compound that has several beneficial effects such as neuroprotection and cytotoxicity for several GBM cell lines. Here we evaluated the mechanism of action of Rsv on human GBM cell lines, focusing on the role of autophagy and its crosstalk with apoptosis and cell cycle control. We further evaluated the role of autophagy and the effect of Rsv on GBM Cancer Stem Cells (gCSCs), involved in GBM resistance and recurrence. Glioma cells treated with Rsv was tested for autophagy, apoptosis, necrosis, cell cycle and phosphorylation or expression levels of key players of these processes. Rsv induced the formation of autophagosomes in three human GBM cell lines, accompanied by an upregulation of autophagy proteins Atg5, beclin-1 and LC3-II. Inhibition of Rsv-induced autophagy triggered apoptosis, with an increase in Bax and cleavage of caspase-3. While inhibition of apoptosis or autophagy alone did not revert Rsv-induced toxicity, inhibition of both processes blocked this toxicity. Rsv also induced a S-G2/M phase arrest, accompanied by an increase on levels of pCdc2(Y15), cyclin A, E and B, and pRb (S807/811) and a decrease of cyclin D1. Interestingly, this arrest was dependent on the induction of autophagy, since inhibition of Rsv-induced autophagy abolishes cell cycle arrest and returns the phosphorylation of Cdc2(Y15) and Rb(S807/811), and levels of cyclin A, and B to control levels. Finally, inhibition of autophagy or treatment with Rsv decreased the sphere formation and the percentage of CD133 and OCT4-positive cells, markers of gCSCs. In conclusion, the crosstalk among autophagy, cell cycle and apoptosis, together with the biology of gCSCs, has to be considered in tailoring pharmacological interventions aimed to reduce glioma growth using compounds with multiple targets such as Rsv.
When autophagy is induced, ULK1 phosphorylates AMBRA1, releasing the autophagy core complex from the cytoskeleton and allowing its relocalization to the ER membrane to nucleate autophagosome formation.
Autophagy is an evolutionary conserved catabolic process involved in several physiological and pathological processes such as cancer and neurodegeneration. Autophagy initiation signaling requires both the ULK1 kinase and the BECLIN 1–VPS34 core complex to generate autophagosomes, double-membraned vesicles that transfer cellular contents to lysosomes. In this study, we show that the BECLIN 1–VPS34 complex is tethered to the cytoskeleton through an interaction between the BECLIN 1–interacting protein AMBRA1 and dynein light chains 1/2. When autophagy is induced, ULK1 phosphorylates AMBRA1, releasing the autophagy core complex from dynein. Its subsequent relocalization to the endoplasmic reticulum enables autophagosome nucleation. Therefore, AMBRA1 constitutes a direct regulatory link between ULK1 and BECLIN 1–VPS34, which is required for core complex positioning and activity within the cell. Moreover, our results demonstrate that in addition to a function for microtubules in mediating autophagosome transport, there is a strict and regulatory relationship between cytoskeleton dynamics and autophagosome formation.
Recently, lipid droplets have been found to be involved in an important cytoplasmic organelle for hepatitis C virus (HCV) production. However, the mechanisms of HCV assembly, budding, and release remain poorly understood. Retroviruses and some other enveloped viruses require an endosomal sorting complex required for transport (ESCRT) components and their associated proteins for their budding process.
To determine whether or not the ESCRT system is needed for HCV production, we examined the infectivity of HCV or the Core levels in culture supernatants as well as HCV RNA levels in HuH-7-derived RSc cells, in which HCV-JFH1 can infect and efficiently replicate, expressing short hairpin RNA or siRNA targeted to tumor susceptibility gene 101 (TSG101), apoptosis-linked gene 2 interacting protein X (Alix), Vps4B, charged multivesicular body protein 4b (CHMP4b), or Brox, all of which are components of the ESCRT system. We found that the infectivity of HCV in the supernatants was significantly suppressed in these knockdown cells. Consequently, the release of the HCV Core into the culture supernatants was significantly suppressed in these knockdown cells after HCV-JFH1 infection, while the intracellular infectivity and the RNA replication of HCV-JFH1 were not significantly affected. Furthermore, the HCV Core mostly colocalized with CHMP4b, a component of ESCRT-III. In this context, HCV Core could bind to CHMP4b. Nevertheless, we failed to find the conserved viral late domain motif, which is required for interaction with the ESCRT component, in the HCV-JFH1 Core, suggesting that HCV Core has a novel motif required for HCV production.
These results suggest that the ESCRT system is required for infectious HCV production.
High doses of ionizing radiation result in biological damage; however, the precise relationships between long-term health effects, including cancer, and low-dose exposures remain poorly understood and are currently extrapolated using high-dose exposure data. Identifying the signaling pathways and individual proteins affected at the post-translational level by radiation should shed valuable insight into the molecular mechanisms that regulate dose-dependent responses to radiation.
We have identified 7117 unique phosphopeptides (2566 phosphoproteins) from control and irradiated (2 and 50 cGy) primary human skin fibroblasts 1 h post-exposure. Semi-quantitative label-free analyses were performed to identify phosphopeptides that are apparently altered by radiation exposure. This screen identified phosphorylation sites on proteins with known roles in radiation responses including TP53BP1 as well as previously unidentified radiation-responsive proteins such as the candidate tumor suppressor SASH1. Bioinformatic analyses suggest that low and high doses of radiation affect both overlapping and unique biological processes and suggest a role for MAP kinase and protein kinase A (PKA) signaling in the radiation response as well as differential regulation of p53 networks at low and high doses of radiation.
Our results represent the most comprehensive analysis of the phosphoproteomes of human primary fibroblasts exposed to multiple doses of ionizing radiation published to date and provide a basis for the systems-level identification of biological processes, molecular pathways and individual proteins regulated in a dose dependent manner by ionizing radiation. Further study of these modified proteins and affected networks should help to define the molecular mechanisms that regulate biological responses to radiation at different radiation doses and elucidate the impact of low-dose radiation exposure on human health.
Autophagy is an evolutionarily conserved protein degradation pathway. A defect in autophagy may contribute to tumorigenesis. Autophagy inducers could have a potential function in tumor prevention and treatment.
Our results showed that Rhabdastrellic acid-A, an isomalabaricane triterpenoid isolated from the sponge Rhabdastrella globostellata, inhibited proliferation of human cancer cell lines Hep3B and A549 and induced caspase-independent cell death in both the cell lines. Further investigation showed that Rhabdastrellic acid-A induced autophagy of cancer cells determined by YFP-LC3 punctation and increased LC3-II. The pretreatment with autophagy inhibitor 3-MA inhibited Rhabdastrellic acid-A-induced cell death. Knockdown of autophagy-related gene Atg5 inhibited Rhabdastrellic acid-A-induced cell death in A549 cells. Also, phospho-Akt and its downstream targets significantly decreased after treatment with Rhabdastrellic acid-A in both cancer cell lines. Transfection of constitutive active Akt plasmid abrogated autophagy and cell death induced by Rhabdastrellic acid-A.
These results suggest that Rhabdastrellic acid-A could induce autophagy-associated cell death through blocking Akt pathway in cancer cells. It also provides the evidence that Rhabdastrellic acid-A deserves further investigation as a potential anticancer or cancer preventive agent.
Spermatogonia- stem cells and progenitors of adult spermatogenesis- are killed through a p53-regulated apoptotic process after γ-irradiation but the death effectors are still poorly characterized. Our data demonstrate that both intrinsic and extrinsic apoptotic pathways are involved, and especially that spermatogonia can be split into two main populations, according to apoptotic effectors. Following irradiation both Dr5 and Puma genes are upregulated in the α6-integrin-positive Side Population (SP) fraction, which is highly enriched in spermatogonia. Flow cytometric analysis confirms an increased number of Dr5-expressing SP cells, and Puma-β isoform accumulates in α6-integrin positive cellular extracts, enriched in spermatogonia. Trail−/− or Puma−/− spermatogonia display a reduced sensitivity to radiation-induced apoptosis. The TUNEL kinetics strongly suggest that the extrinsic and intrinsic pathways, via Trail/Dr5 and Puma respectively, could be engaged in distinct subpopulations of spermatogonia. Indeed flow cytometric studies show that Dr5 receptor is constitutively present on more than half of the undifferentiated progenitors (Kit− α6+ SP) and half of the differentiated ones (Kit+ α6+ SP). In addition after irradiation, Puma is not detected in the Dr5-positive cellular fraction isolated by immunomagnetic purification, while Puma is present in the Dr5-negative cell extracts. In conclusion, adult testicular progenitors are divided into distinct sub-populations by apoptotic effectors, independently of progenitor types (immature Kit-negative versus mature Kit-positive), underscoring differential radiosensitivities characterizing the stem cell/progenitors compartment.
Interferon beta (IFNβ) is the most common immunomodulatory treatment for relapsing-remitting multiple sclerosis (RRMS). However, some patients fail to respond to treatment. In this study, we identified putative clinical response markers in the serum and plasma of people with multiple sclerosis (MS) treated with IFNβ. In a discovery-driven approach, we use 2D-difference gel electrophoresis (DIGE) to identify putative clinical response markers and apply power calculations to identify the sample size required to further validate those markers. In the process we have optimized a DIGE protocol for plasma to obtain cost effective and high resolution gels for effective spot comparison. APOA1, A2M, and FIBB were identified as putative clinical response markers. Power calculations showed that the current DIGE experiment requires a minimum of 10 samples from each group to be confident of 1.5 fold difference at the p<0.05 significance level. In a complementary targeted approach, Cytometric Beadarray (CBA) analysis showed no significant difference in the serum concentration of IL-6, IL-8, MIG, Eotaxin, IP-10, MCP-1, and MIP-1α, between clinical responders and non-responders, despite the association of these proteins with IFNβ treatment in MS.
Chemotherapy-induced reduction in tumor load is a function of apoptotic cell death, orchestrated by intracellular caspases. However, the effectiveness of these therapies is compromised by mutations affecting specific genes, controlling and/or regulating apoptotic signaling. Therefore, it is desirable to identify novel pathways of cell death, which could function in tandem with or in the absence of efficient apoptotic machinery. In this regard, recent evidence supports the existence of a novel cell death pathway termed autophagy, which is activated upon growth factor deprivation or exposure to genotoxic compounds. The functional relevance of this pathway in terms of its ability to serve as a stress response or a truly death effector mechanism is still in question; however, reports indicate that autophagy is a specialized form of cell death under certain conditions.
We report here the simultaneous induction of non-canonical autophagy and apoptosis in human cancer cells upon exposure to a small molecule compound that triggers intracellular hydrogen peroxide (H2O2) production. Whereas, silencing of beclin1 neither inhibited the hallmarks of autophagy nor the induction of cell death, Atg 7 or Ulk1 knockdown significantly abrogated drug-induced H2O2-mediated autophagy. Furthermore, we provide evidence that activated extracellular regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) are upstream effectors controlling both autophagy and apoptosis in response to elevated intracellular H2O2. Interestingly, inhibition of JNK activity reversed the increase in Atg7 expression in this system, thus indicating that JNK may regulate autophagy by activating Atg7. Of note, the small molecule compound triggered autophagy and apoptosis in primary cells derived from patients with lymphoma, but not in non-transformed cells.
Considering that loss of tumor suppressor beclin 1 is associated with neoplasia, the ability of this small molecule compound to engage both autophagic and apoptotic machineries via ROS production and subsequent activation of ERK and JNK could have potential translational implications.
Protein kinases are major components of signal transduction pathways in multiple cellular processes. Kinases directly interact with and phosphorylate downstream substrates, thus modulating their functions. Despite the importance of identifying substrates in order to more fully understand the signaling network of respective kinases, efficient methods to search for substrates remain poorly explored.
We combined mass spectrometry and affinity column chromatography of the catalytic domain of protein kinases to screen potential substrates. Using the active catalytic fragment of Rho-kinase/ROCK/ROK as the model bait, we obtained about 300 interacting proteins from the rat brain cytosol fraction, which included the proteins previously reported as Rho-kinase substrates. Several novel interacting proteins, including doublecortin, were phosphorylated by Rho-kinase both in vitro and in vivo.
This method would enable identification of novel specific substrates for kinases such as Rho-kinase with high sensitivity.